Method for detecting varicella-zoster virus gE protein specific IgG antibody in human serum by high-sensitivity ELISA (Enzyme-Linked Immunosorbent Assay) method

Abstract

The invention relates to the technical field of biology, in particular to a method for detecting a varicella-zoster virus gE protein specific IgG antibody in human serum by a high-sensitivity ELISA (Enzyme-Linked Immunosorbent Assay) method.A recombinant VZV-gE protein is adopted to coat an elisa plate, a self-established indirect ELISA method is adopted, and the specific IgG antibody of the varicella-zoster virus gE protein in the human serum is detected by screening the coating concentration, the closing condition, the incubation time, the secondary antibody concentration and the like. The method for detecting the concentration of the VZV-gE protein IgG antibody in the human serum is successfully established; then the positive mixed human serum is calibrated by using the method and an NIBSC international standard substance (NIBSC code: W1044), and the calibrated positive mixed serum is used as an enterprise standard substance for determining the concentration of a VZV-gE protein specific IgG antibody in the human serum; in this way, the technical problem that in the prior art, a detection method for the varicella-zoster virus gE protein specific IgG antibody in human serum cannot meet the requirements for high sensitivity, excellent specificity, high flux and excellent reproducibility at the same time is solved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting varicella-zoster virus gE protein-specific IgG antibodies in human serum by a high-sensitivity ELISA method. Background Art

[0002] Varicella-zoster virus (VZV) is the pathogen that causes chickenpox and herpes zoster in humans. It belongs to the family of Herpesviridae and is a double-stranded DNA virus. VZV virus particles are quasi-spherical with a diameter of 150 to 200 nm. They are divided into four layers from the inside to the outside, namely the virus core, nucleocapsid, cortex and envelope. The viral envelope contains at least 9 glycoproteins, including gE, gB, gH, gI, gC, gL, gK, gM and gN, which play an important role in the virus infecting the host.

[0003] The most effective way to avoid infection with the virus is vaccination. Currently, VZV vaccines mainly include attenuated vaccines and recombinant protein vaccines, and nucleic acid vaccines are also under development. In 1974, Japan's Takahashi Rinaki established the Oka strain of varicella virus. The World Health Organization believes that currently only the Oka strain is suitable for the preparation of live attenuated varicella vaccine. Countries around the world use the Oka strain of live attenuated varicella vaccine to prevent and control varicella. Studies have shown that the biological characteristics of the Oka strain remain well-stable after being passed down to the 50th generation.

[0004] At present, the vaccines used to prevent herpes zoster virus are mainly GSK's subunit vaccine (SHINGRIX) and Changchun Bio-Tech's live attenuated vaccine. SHINGRIX vaccine is composed of the gE glycoprotein with the strongest antigenicity and the highest content in VZV and the AS01B adjuvant system, which can stimulate the body's strong cellular immunity and humoral immunity. By looking at the FDA's Review of SHINGRIX, in terms of vaccine immunogenicity evaluation, it mainly focuses on the Anti-gE antibody level (ELISA) and gE-specific cytokine level (ICS). These two indicators are used as the main endpoint indicators of the vaccine's immunogenicity effect. In one of its Phase III clinical trials, GSK established an ELISA method with a cut-off value of 97mIU / mL (i.e., the positive conversion judgment threshold) to detect its clinical samples. At the same time, in terms of cellular immunity evaluation, GSK evaluated the proportion of CD4+ cells in biological samples that met at least two specific cytokine levels, i.e., the frequencies of glycoprotein E-specific CD4[2+]T cells. The cut-off is 320CD4[2+]T cells per 10 6CD4 T cells were counted.

[0005] By checking CDE's "Technical Review Report on GSK's Application for Marketing of Recombinant Herpes Zoster Vaccine (JXSS1800039)", it can be found that CDE does not support GSK's use of Anti-gE antibody levels (ELISA) to directly infer protective efficacy, and it can only be used for reference; therefore, the FAMA method, as the "gold standard" for varicella-specific antibody detection, is increasingly receiving attention from regulators in China.

[0006] The glycoprotein antibodies that are mainly focused on in the study of the immunogenicity of the varicella-zoster virus vaccine are gE protein antibodies, and some researchers have also tested gB and gH protein antibodies. The FAMA method can detect all specific IgG antibodies against VZV, and its sensitivity and specificity are better than those of the ELISA method. However, the FAMA method has a more complicated detection process, low throughput, and more difficult reproducibility than ELISA. Therefore, domestic clinical trials generally use both methods to evaluate the humoral immunity of the vaccine, and regulatory agencies also use clinical trial data to evaluate the correlation between the two methods in order to evaluate the immunogenicity of the vaccine.

[0007] The established ELISA method for detecting the level of gE antigen-specific antibodies uses NIBSC international standards to prepare standard curve samples and quality control samples. If it is a non-clinical evaluation, positive serum or positive antibodies are required; the recombinant VZV-gE protein is coated on the ELISA plate, and after blocking, the standard, quality control and test samples are added for incubation, and the anti-human IgG secondary antibody is added again. Finally, the plate is washed and TMB is used for color development, and the VZV-gE protein-specific IgG antibody concentration in the test sample is calculated using the international standard.

[0008] The FAMA method also requires the use of fixing cells infected with VZV virus on a glass slide, adding standard substances and test samples for incubation, adding fluorescent secondary antibodies and observing under a microscope. The wells with fluorescent rings are positive wells. The results can be determined based on the test results of a certain number of negative serum wells. Clinically, it is generally judged as positive when ≥1:4, that is, it has VZV protection.

[0009] In terms of methodological validation, the current literature mainly focuses on the establishment of methodology but lacks systematic methodological validation; the China Food and Drug Inspection Institute has published literature on the preparation and validation of VZV IgG antibody national standards, which uses NIBSC international standards to assign values ​​to the positive reference materials of the Institute to evaluate the performance of VZV-related in vitro diagnostic kits; Zhou Zhijun et al. used commercial kits to validate the VZV virus IgG detection method, and the main contents of the validation included linearity and linear range, precision and accuracy, and addition tests, but the validation indicators could not meet the requirements for clinical trial submission; according to ICH M10 and the 2020 edition of the "Chinese Pharmacopoeia" require that the validation of ligand binding quantitative analysis methods should at least include calibration curves and ranges, accuracy and precision, specificity, selectivity, dilution linearity and hook effect, stability, etc., and ISR should also be performed on samples; while qualitative antibody detection methods shall be carried out in accordance with the "Technical Guidelines for Research on Antibody Analysis Methods for Vaccine Clinical Trials (Draft for Comments)" for cut-off values, specificity, and detection limits, and quantitative and semi-quantitative detection methods shall be validated for specificity, detection limit, linearity, linear range, accuracy and precision.

[0010] There is a lack of national standards for the methodological validation of FAMA. Generally speaking, it can be carried out by referring to relevant literature, but it should generally follow the relevant guidelines for antibody detection. Yuan Dian et al. validated the sensitivity, specificity, repeatability, and accuracy of the established FAMA method, with a sensitivity of 31.25 mIU / mL and GSV% of 29.95% and 26.71%, respectively. At the same time, specificity validation found no cross-reaction with HSV-1 / 2 antibodies, demonstrating good specificity.

[0011] Park R, Hwang JY, Lee KI, etc. considered that FAMA requires P2 laboratory to culture the virus and tried to establish a FAMA based on gE protein, but this method is difficult to obtain regulatory approval; Liu JJ, etc. used FAMA to analyze and detect 592 sera in the laboratory, but after the method was established, only the sensitivity and specificity were verified; Michalik DE, etc. used ELISA and FAMA to detect serum samples from 148 children in clinical trials, but did not describe the FAMA methodological validation indicators.

[0012] In summary, the existing detection methods for IgG antibodies specific to varicella-zoster virus gE protein in human serum cannot simultaneously meet the requirements of high sensitivity, excellent specificity, high throughput, and excellent reproducibility. Summary of the invention

[0013] The object of the present invention is to provide a method for detecting varicella-zoster virus gE protein-specific IgG antibodies in human serum by a high-sensitivity ELISA method, aiming to solve the technical problem that the detection method for varicella-zoster virus gE protein-specific IgG antibodies in human serum in the prior art cannot simultaneously meet the requirements of high sensitivity, excellent specificity, high throughput and excellent reproducibility.

[0014] To achieve the above object, the present invention adopts a high-sensitivity ELISA method for detecting IgG antibodies specific to varicella-zoster virus gE protein in human serum, comprising the following steps:

[0015] Antigen coating: dilute the gE protein with a content of 2170 μg / mL to 0.5 μg / mL with 1× PBS phosphate buffer, add 100 μL / well to a 96-well plate, and keep at low temperature overnight;

[0016] Washing the plate: Take out the ELISA plate coated overnight, discard the liquid in the well, pat dry, add 300μL / well of washing solution, let it stand and pat dry, repeat three times;

[0017] Blocking: Add 300 μL / well of blocking solution, cover with sealing film and incubate;

[0018] Repeat the plate washing process;

[0019] Sample addition: Add 100 μL / well of the prepared quality control sample, cover with sealing film and incubate;

[0020] Repeat the plate washing process;

[0021] Add enzyme: add 100 μL / well of enzyme working solution, cover with sealing film and incubate;

[0022] Repeat the plate washing process;

[0023] Color development: TMB 100 μL / well was added, and the plate was covered with a sealing film to avoid light;

[0024] Stop: Add 50 μL / well of stop solution and shake gently;

[0025] Reading: Read the OD value at a wavelength of 450nm.

[0026] Among them, the low temperature overnight is 2-8℃.

[0027] Among them, take out the ELISA plate coated overnight, discard the liquid in the wells, pat dry, add 300 μL / well of washing solution, let it stand and pat dry, and the standing time is 1 minute.

[0028] Among them, 300 μL / well of blocking solution was added, and the plate was covered with a sealing film and incubated at 37° C. and 400 rpm for 2 hours.

[0029] Among them, 100 μL / well of the prepared quality control sample was added, covered with a sealing film and incubated at 37°C and 400 rpm for 1 hour.

[0030] Among them, 100 μL / well of the enzyme working solution was added, and the plate was covered with a sealing film and incubated at 37° C. and 400 rpm for 30 minutes.

[0031] Among them, 100 μL / well of TMB was added, the plate was covered with a sealing film and the color was developed in the dark at 37° C. and 400 rpm for 15 minutes.

[0032] The invention discloses a method for detecting varicella-zoster virus gE protein-specific IgG antibody in human serum by a high-sensitivity ELISA method. The method adopts a recombinant VZV-gE protein to coat an enzyme label plate, and a self-constructed indirect ELISA method is used. By screening the coating concentration, blocking conditions, incubation time, secondary antibody concentration, etc., a method for detecting the concentration of VZV-gE protein IgG antibody in human serum is successfully established; then the method and NIBSC international standard (NIBSC code: W1044) are used to calibrate positive mixed human serum, and the calibrated positive mixed serum is used as an enterprise standard to determine the concentration of VZV-gE protein-specific IgG antibody in human serum;

[0033] In this way, the technical problem that the detection method for varicella-zoster virus gE protein-specific IgG antibodies in human serum in the prior art cannot simultaneously meet the requirements of high sensitivity, excellent specificity, high throughput and excellent reproducibility is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 The invention is a flowchart of the steps of a method for detecting IgG antibodies specific to varicella-zoster virus gE protein in human serum by a high-sensitivity ELISA method. DETAILED DESCRIPTION

[0036] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0037] See also Figure 1 , Figure 1 The invention is a flowchart of the steps of a method for detecting IgG antibodies specific to varicella-zoster virus gE protein in human serum by a high-sensitivity ELISA method.

[0038] The present invention provides a method for detecting IgG antibodies specific to varicella-zoster virus gE protein in human serum by high-sensitivity ELISA method, comprising the following steps:

[0039] Antigen coating: dilute the gE protein with a content of 2170 μg / mL to 0.5 μg / mL with 1× PBS phosphate buffer, add 100 μL / well to a 96-well plate, and keep at low temperature overnight;

[0040] Washing the plate: Take out the ELISA plate coated overnight, discard the liquid in the well, pat dry, add 300μL / well of washing solution, let it stand and pat dry, repeat three times;

[0041] Blocking: Add 300 μL / well of blocking solution, cover with sealing film and incubate;

[0042] Repeat the plate washing process;

[0043] Sample addition: Add 100 μL / well of the prepared quality control sample, cover with sealing film and incubate;

[0044] Repeat the plate washing process;

[0045] Add enzyme: add 100 μL / well of enzyme working solution, cover with sealing film and incubate;

[0046] Repeat the plate washing process;

[0047] Color development: TMB 100 μL / well was added, and the plate was covered with a sealing film to avoid light;

[0048] Stop: Add 50 μL / well of stop solution and shake gently;

[0049] Reading: Read the OD value at a wavelength of 450nm.

[0050] The overnight temperature is 2-8℃.

[0051] Take out the ELISA plate coated overnight, discard the liquid in the wells, pat dry, add 300 μL / well of wash solution, let it stand and pat dry, the standing time is 1 min.

[0052] 300 μL / well of blocking solution was added, and the plate was covered with a sealing film and incubated at 37°C and 400 rpm for 2 h.

[0053] Add 100 μL / well of the prepared quality control sample, cover with sealing film and incubate at 37°C and 400 rpm for 1 hour.

[0054] 100 μL / well of enzyme working solution was added, and the plate was covered with sealing film and incubated at 37°C and 400 rpm for 30 min.

[0055] TMB 100 μL / well was added, and the plate was covered with a sealing film to protect from light. The color development was carried out in the dark at 37°C and 400 rpm for 15 min.

[0056] According to this specific embodiment, the method uses a recombinant VZV-gE protein to coat an ELISA plate, and establishes an indirect ELISA method. By screening the coating concentration, blocking conditions, incubation time, secondary antibody concentration, etc., a method for detecting the concentration of VZV-gE protein IgG antibodies in human serum is successfully established; then the method and the NIBSC international standard (NIBSC code: W1044) are used to calibrate the positive mixed human serum, and the calibrated positive mixed serum is used as an enterprise standard to determine the concentration of VZV-gE protein-specific IgG antibodies in human serum;

[0057] In this way, the technical problem that the detection method for varicella-zoster virus gE protein-specific IgG antibodies in human serum in the prior art cannot simultaneously meet the requirements of high sensitivity, excellent specificity, high throughput and excellent reproducibility is solved.

[0058] At the same time, the present invention performs methodological verification on the established ELISA method for detecting the concentration of VZV-gE protein-specific IgG antibodies in human serum according to ICH M10;

[0059] The content of methodological validation includes: standard curve and quantitative range, precision and accuracy, method durability, lipemia selectivity, hemolysis selectivity, room temperature stability, 2-8℃ refrigerator stability, freeze-thaw stability, method durability, long-term stability of samples, etc.

[0060] Dilute the recombinant VZV-gE protein to 0.5μg / mL with 1×PBS phosphate buffer, add 100μL / well to a 96-well plate, and place at 2-8℃ for 16-18h. The next day, take out the plate washer and add 300μL / well of blocking solution, place at 37℃, 400rpm for blocking for 2h. After the blocking is completed, add 100μL / well of the prepared standard curve, quality control, and the sample to be tested to the ELISA plate, place at 37℃, 400rpm for reaction for 1h. After the reaction is completed, wash the plate, add 100μL / well of the detection antibody working solution and place at 37℃, 400rpm for reaction for 30min. After washing the plate 5 times, add 100μL / well of the single-component TMB colorimetric solution, place at 37℃, 400rpm for color development in the dark for 15min. Finally, use a full-wavelength microplate reader to read the signal value at OD450nm. Use the software provided by the microplate reader to perform four-parameter fitting to obtain the concentration of the sample to be tested.

[0061] The specific methods of method validation are as follows:

[0062] Standard curve and quantitation range validation:

[0063] Take positive mixed human serum and use artificial serum / plasma II as diluent to prepare 8 concentration samples from SA to SH. Prepare them as needed. The preparation method is shown in Table 4. The precision and accuracy need to be determined in at least 6 analytical batches, which must be completed by at least 2 analysts in no less than 3 days.

[0064]

[0065] Table 1

[0066] Precision and Accuracy Verification:

[0067] Take positive mixed human serum and use artificial serum / plasma II as diluent to prepare 8 concentration samples from SA to SH; take positive mixed human serum and use negative human serum as diluent to prepare 5 concentration samples of quality control samples ULOQ, QH, QM, QL, LLOQ, and prepare 3 groups in parallel; precision and accuracy need to be determined in at least 6 analytical batches, and at least 2 analysts need to complete it in no less than 3 days;

[0068]

[0069] Table 2

[0070] Lipidemia Selective Validation:

[0071] Triglyceride (50 mg / mL) solution: weigh triglyceride and add appropriate amount of acetonitrile to dissolve it to a concentration of 50 mg / mL;

[0072] Lipemic serum (5 mg / mL): dilute 50 mg / mL triglyceride solution to 5 mg / mL with artificial serum / plasma II;

[0073] Using lipemic serum as diluent, three groups of samples were prepared in parallel at ULOQ to LLOQ. The lipemic serum without analyte diluted 100 times was used as lipemic blank control to investigate the selectivity of lipemic serum.

[0074]

[0075] Table 3

[0076] Solution preparation method: 1×PBS: Take 1mL of 20×PBS phosphate buffer, add it to 19mL of ultrapure water, and mix well; valid for the same day;

[0077] Coating solution (0.5 μg / mL): Take 3 μL gE protein, add 13017 μL 1× PBS phosphate buffer, mix well; prepare and use immediately;

[0078] Washing solution (1×PBST solution): Measure 100 mL of 20×PBS and pour it into a 2000 mL glass bottle, add 1900 mL of ultrapure water, add 1 mL of Tween-20, mix well, and store in a refrigerator at 2-8°C. The validity period is 1 month.

[0079] Blocking solution: Take 6 mL of blocking solution III, add it to 24 mL of ultrapure water, mix well and the solution is valid for the same day.

[0080] Sample diluent: Take 5 mL of sample diluent I, add it to 15 mL of ultrapure water, mix well and the sample is valid for the same day.

[0081] Negative sample (NC): Take 10 μL of negative human serum, add it to 990 μL of artificial serum / plasma II, mix well, and it is valid on the same day;

[0082] Preparation of enzyme conjugate working solution: (1) Take rabbit anti-human IgG H&L (HRP) and dilute it 100 times with PBST (washing solution), mix well, and obtain enzyme conjugate intermediate working solution A; (2) Take enzyme conjugate intermediate working solution A and dilute it 1000 times with PBST (washing solution), mix well, and obtain enzyme conjugate intermediate working solution A. Prepare it immediately before use and it is valid on the same day.

[0083] Stop solution (2M H2SO4): Measure 110mL of concentrated sulfuric acid and slowly add it into 890mL of ultrapure water while stirring continuously. Use after cooling to room temperature and store at room temperature. The shelf life is 3 months.

[0084] Room temperature stability verification:

[0085] Room temperature stability samples: See "Solution preparation method" for preparation method; Use high (Y1) and low (Y2) concentrations of quality control samples to simultaneously conduct room temperature stability tests, and prepare and test three samples at each concentration and each test time point. The test time points are 2h, 4h, and 24h, with one sample for each time point. Prepare room temperature stability samples and store them in a -80℃ refrigerator; When validating the method, take them out 24h, 4h, and 2h in advance and place them at room temperature (the specific placement time is subject to actual records), and dilute them 100 times with blank human serum / plasma II before adding samples; Treat the stability samples at room temperature according to the established method and then test them;

[0086] Refrigerator Stability Verification:

[0087] See "Solution Preparation Method" for preparation method; high (Y1) and low (Y2) concentrations of quality control samples were used to simultaneously conduct stability tests in a 2-8°C refrigerator. Three samples were prepared and tested at each concentration and each test time point; the test time points were 2h, 4h, and 24h, with one sample for each time point. The stability samples were prepared and stored in a -80°C refrigerator. When validating the method, the samples were taken out 24h, 4h, and 2h in advance and placed at room temperature (the specific placement time is subject to actual records), and diluted 100 times with blank human serum / plasma II before adding the sample; the stability samples placed in a 2-8°C refrigerator were treated according to the established method and tested.

[0088] Freeze-thaw stability verification:

[0089] See “Solution Preparation Method” for preparation method; use high (Y1) and low (Y2) concentrations of two quality control samples for freeze-thaw stability testing at the same time, prepare and test three samples for each concentration and each testing number; prepare freeze-thaw stability samples and store them in a -80℃ refrigerator. The testing number is 1, 2, 3, 4, 5 and 6 cycles, one copy for each cycle, each freezing time is at least 12h, and the thawing time at room temperature is not less than 30min. Dilute 100 times with blank human serum / plasma II before adding samples; process freeze-thaw samples according to the established method and then test.

[0090] Sample long-term stability verification:

[0091] See “Solution Preparation Method” for preparation method; use high (Y1) and low (Y2) concentrations of two quality control samples for simultaneous long-term stability study of samples, prepare and measure three samples for each concentration and each study time point; prepare long-term stability samples and store them in a -80°C refrigerator, the study time points are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 months (the window period is ±7 working days, if delayed due to force majeure, record according to the actual time point), one aliquot for each time point, and dilute 100 times with blank human serum / plasma II before adding samples; process long-term samples according to the established method and then test them.

[0092] Method robustness validation:

[0093] (1) Comparison of ELISA instruments: During the methodological validation process, a batch of samples is randomly selected and read on different ELISA instruments.

[0094] (2) Reading time after termination: During the method validation process, a random sample of an analysis batch is selected for analysis. Readings are taken at 10 min, 20 min, and 30 min after termination.

[0095] Hook effect verification:

[0096] Take positive mixed human serum and use artificial serum / plasma II as diluent to prepare HE1, HE2, HE3, and HE4 as hook effect samples;

[0097]

[0098] Table 4

[0099] Blank matrix selectivity verification:

[0100] Take 10 blank human sera from different sources and dilute them 100 times with artificial serum / plasma II as diluents to prepare QH and LLOQ as blank matrix selective samples. The preparation method is shown in Table 5. At the same time, a blank well needs to be set for each source of serum.

[0101]

[0102] Table 5

[0103] Dilution Linearity Verification:

[0104] Artificial serum / plasma II was diluted 100 times as a diluent to prepare XY1, XY2, and XY3 as dilution linear samples. Three samples of each sample were prepared independently.

[0105]

[0106] Table 6

[0107] Specificity Verification:

[0108] Oseltamivir (1000ng / mL) solution: Weigh the oseltamivir reference substance, dissolve it in methanol, and prepare a 500000ng / mL oseltamivir intermediate working solution. Then take 5ul of the oseltamivir intermediate working solution, add it to 2495ul of artificial serum / plasma II, and mix well;

[0109] Interferon α2b (2ug / mL) solution: Take 3ul of interferon α2b stock solution, add it to 3042ul of artificial serum / plasma II, and mix well;

[0110] SARS-CoV-2 S protein antibody (10000RU / mL) solution: Take 200ul of SARS-CoV-2 S protein antibody, add it to 3800ul of artificial serum / plasma II, and mix well;

[0111] Oseltamivir (1000 ng / mL) solution, interferon α2b (2 ug / mL) solution, and new crown S protein antibody (10000 RU / mL) solution were used as diluents to prepare ULOQ and LLOQ as specific samples. Each sample was prepared in parallel in 3 groups. The preparation method is shown in Table 7. At the same time, oseltamivir (1000 ng / mL) solution, interferon α2b (2 ug / mL) solution, and new crown S protein antibody (10000 RU / mL) solution were used as blank samples with related interfering substances added for investigation;

[0112]

[0113] Table 7

[0114] Hemolysis selectivity validation:

[0115] Preparation of hemolyzed samples: Hemolyzed serum (2% V / V): Take 5ul of hemolyzed whole blood or red blood cells, add to 245ul of artificial serum / plasma II, and mix well;

[0116] 100x hemolyzed serum: Take 50ul of hemolyzed serum, add it to 4950ul of artificial serum / plasma II, and mix well;

[0117] 100x hemolytic serum was used as diluent to prepare QH and LLOQ as hemolytic samples, and three groups were prepared in parallel; 100x hemolytic serum was used as hemolytic blank control to investigate the selectivity of hemolytic serum;

[0118]

[0119] Table 8

[0120] Methodology validation results:

[0121] A total of 19 batches were run in this validation, and the information summary of each batch is shown in Appendix Table 9;

[0122] Serial number Analysis batch name Verify content Batch analysis 1 KY-B2304-2-20231130-1.skax Intra-batch / inter-batch precision and accuracy Acceptable 2 KY-B2304-2-20231130-2.skax Intra-batch / inter-batch precision and accuracy Acceptable 3 KY-B2304-2-20231201-2.skax Intra-batch / inter-batch precision and accuracy Acceptable 4 KY-B2304-2-20231205-1.skax Intra-batch / inter-batch precision and accuracy Acceptable 5 KY-B2304-2-20231205-2.skax Intra-batch / inter-batch precision and accuracy Acceptable 6 KY-B2304-2-20231206-1.skax Intra-batch / inter-batch precision and accuracy Acceptable 7 KY-B2304-2-20231218-1.skax Lipemia*, 38-day long-term stability Acceptable 8 KY-B2304-2-20231227-1.skax Lipidemia, hook effect Acceptable 9 KY-B2304-2-20231228-1.skax Stability in refrigerator at 2-8℃ for 24h Acceptable 10 KY-B2304-2-20231228-2.skax Stability at room temperature for 24 hours Acceptable 11 KY-B2304-2-20240115-1.skax Long-term stability 65 days, stability after 2 freeze-thaw cycles Acceptable 12 KY-B2304-2-202401 15-2.skax Stability after 6 freeze-thaw cycles Acceptable 13 KY-B2304-2-20240227-1.skax Long-term stability 107 days Acceptable 14 KY-B2304-2-20240326-1.skax Long-term stability 135 days Acceptable 15 KY-B2304-2-20240411-1.skax Dilution linearity Acceptable 16 KY-B2304-2-20240411-2.skax Blank matrix selectivity Acceptable 17 KY-B2304-2-20240412-1.skax Specificity Acceptable 18 KY-B2304-2-20240425-1.skax Long-term stability 164 days Acceptable 19 KY-B2304-2-20240523-1.skax Long-term stability 191 days, hemolytic selectivity Acceptable

[0123] Table 9: Validation run batch summary

[0124] Note: “*” indicates that the analysis batch does not meet the requirements.

[0125] Precision:

[0126] The precision verification results are shown in Table 10: the maximum intra-batch precision of the upper limit of quantification ULOQ is 8.93%, and the inter-batch precision is 7.43%; the maximum intra-batch precision of the lower limit of quantification LLOQ is 9.84%, and the inter-batch precision is 12.12%; the maximum intra-batch precision of the quality control samples (QH, QM, QL) other than ULOQ and LLOQ is 12.99%, and the maximum inter-batch precision is 11.54%. The precision verification results meet the requirements.

[0127] Accuracy:

[0128] The accuracy verification results are shown in Table 10: the intra-batch accuracy deviation of the upper limit of quantification ULOQ is in the range of -19.36% to 1.01%, and the inter-batch accuracy deviation is -7.10%; the intra-batch accuracy deviation of the lower limit of quantification LLOQ is in the range of -16.16% to 16.42%, and the inter-batch accuracy deviation is 5.60%; the intra-batch accuracy deviation of the quality control samples (QH, QM, QL) other than ULOQ and LLOQ is in the range of -17.89% to 13.93%, and the inter-batch accuracy deviation is in the range of -6.72% to -2.50%. The accuracy verification results meet the requirements.

[0129]

[0130]

[0131]

[0132] Table 10: Intra-batch and inter-batch precision and accuracy of quality control samples

[0133] Standard curve and quantitation range:

[0134] The verification results of the standard curve and quantitative range are shown in Tables 11 and 12: In the 8 analysis batches, R2 meets ≥0.98. The accuracy deviation of each concentration of the standard curve in the quantitative range of 0.3125 to 40 mIU / mL is within the range of -12.96% to 19.17%. The verification results of the standard curve and quantitative range meet the requirements.

[0135]

[0136]

[0137] Table 11: Summary of standard curve parameters

[0138]

[0139] Table 12: Back-calculated concentration table of standard curve

[0140] Lipidemia selectivity:

[0141] The results of the lipemia selectivity validation are shown in Table 13: In the lipemia samples prepared with lipemia serum (containing 5 mg / mL of triglycerides) as the diluent, the LLOQ concentration sample accuracy deviation is -19.65%, the ULOQ concentration sample accuracy deviation is 4.97%, the blank serum OD value is lower than the quantification lower limit OD value, and 93.33% of the samples are within the accuracy range of ±20% (LLOQ is ±25%). And the detection OD value of the matrix without the addition of the analyte should be lower than the quantification lower limit OD value. The results of the lipemia selectivity validation meet the requirements.

[0142]

[0143] Table 13: Summary of lipemia selective experimental data

[0144] Room temperature stability:

[0145] The room temperature stability verification results are shown in Table 14: After 2 hours at room temperature, 100% of the QH and QL concentration level quality control samples had a back-calculated concentration within the ±20% range of the labeled value; after 4 hours at room temperature, 83.33% of the QH and QL concentration level quality control samples had a back-calculated concentration within the ±20% range of the labeled value; after 24 hours at room temperature, 100% of the QH and QL concentration level quality control samples had a back-calculated concentration within the ±20% range of the labeled value. The samples were stable after 24 hours at room temperature.

[0146]

[0147] Table 14: Room temperature stability test data summary

[0148] Note: Data with “*” do not meet the acceptance criteria (within ±20%).

[0149] 2-8℃ refrigerator storage stability:

[0150] The results of stability verification in a 2-8℃ refrigerator are shown in Table 15: After being placed in a 2-8℃ refrigerator for 2 hours, 100% of the QH and QL concentration level quality control samples had a back-calculated concentration within the ±20% range of the labeled value; after being placed in a 2-8℃ refrigerator for 4 hours, 100% of the QH and QL concentration level quality control samples had a back-calculated concentration within the ±20% range of the labeled value; after being placed in a 2-8℃ refrigerator for 24 hours, 83.33% of the QH and QL concentration level quality control samples had a back-calculated concentration within the ±20% range of the labeled value. The sample was stable after being placed in a 2-8℃ refrigerator for 24 hours.

[0151]

[0152] Table 15: Summary of 2-8℃ refrigerator storage stability test data

[0153] Note: Data with “*” do not meet the acceptance criteria (within ±20%).

[0154] Freeze-thaw stability:

[0155] The freeze-thaw stability verification results are shown in Table 16: When the samples were frozen and thawed once at -80°C, the back-calculated concentrations of 100% of the QH and QL concentration level quality control samples were within the ±20% range of the labeled value; after freezing and thawing twice, the back-calculated concentrations of 100% of the QH and QL concentration level quality control samples were within the ±20% range of the labeled value; after freezing and thawing three times, the back-calculated concentrations of 100% of the QH and QL concentration level quality control samples were within the ±20% range of the labeled value; after freezing and thawing four times, the back-calculated concentrations of 100% of the QH and QL concentration level quality control samples were within the ±20% range of the labeled value. After freezing and thawing for 5 times, the back-calculated concentrations of 83.33% of the quality control samples at QH and QL concentration levels were within the range of ±20% of the labeled value; after freezing and thawing for 5 times, the back-calculated concentrations of 83.33% of the quality control samples at QH and QL concentration levels were within the range of ±20% of the labeled value; after freezing and thawing for 6 times, the back-calculated concentrations of 100% of the quality control samples at QH and QL concentration levels were within the range of ±20% of the labeled value; the samples were repeatedly frozen and thawed for 6 times under -80℃ conditions, and the results of stability verification met the requirements.

[0156]

[0157] Table 16: Summary of freeze-thaw stability test data

[0158] Long-term sample stability:

[0159] The results of long-term stability verification of the samples are shown in Table 17: after the samples were frozen at -80°C for 38 days, the back-calculated concentrations of 100% of the QH and QL concentration level quality control samples were within the range of ±20% of the labeled value; after 65 days of freezing, the back-calculated concentrations of 100% of the QH and QL concentration level quality control samples were within the range of ±20% of the labeled value; after 107 days of freezing, the back-calculated concentrations of 100% of the QH and QL concentration level quality control samples were within the range of ±20% of the labeled value; after 135 days of freezing, the back-calculated concentrations of 100% of the QH and QL concentration level quality control samples were within the range of ±20% of the labeled value; after 164 days of freezing, the back-calculated concentrations of 100% of the QH and QL concentration level quality control samples were within the range of ±20% of the labeled value; the samples can be frozen for 164 days at -80°C. After 191 days of cryopreservation, 100% of the QH and QL concentration level quality control samples had calculated concentrations within ±20% of the labeled value; samples can be cryopreserved for 191 days at -80°C.

[0160]

[0161]

[0162] Table 17: Summary of long-term stability test data of samples

[0163] Method Durability:

[0164] The verification results of method durability are shown in Table 18: ① For the same analysis batch, readings were taken on different microplate readers, and both the standard curve and quality control samples met the requirements. ② Readings were taken immediately after termination, at 10 minutes, and at 20 minutes, and both the standard curve and quality control samples met the requirements. When readings were taken 30 minutes after termination, the LLOQ of the quality control samples did not meet the requirements. Therefore, readings should be taken within 20 minutes after termination.

[0165]

[0166] Table 18: Summary Table of Method Durability Experiment Data

[0167] Hook effect:

[0168] The verification results of the hook effect are shown in Table 19: OD values of hook effect samples: OD(HE5) < QD(HE6) < OD(HE7) < OD(HE8). There is no hook effect in this detection method.

[0169]

[0170] Table 19: Summary Table of Hook Effect Experiment Data

[0171] Blank matrix selectivity:

[0172] The verification results of blank matrix selectivity are shown in Table 20: For quality control samples prepared from 10 blank sera from different sources, among 100% of different sources, the accuracy of LLOQ should be within ±25% of the labeled value, and the accuracy of QH should be within ±20% of the labeled value. The OD values of 100% of blank samples from different sources should be lower than the OD value of LLOQ. The verification results of blank matrix selectivity meet the requirements.

[0173]

[0174]

[0175] Table 20: Summary Table of Blank Matrix Selectivity Experiment Data

[0176] Dilution linearity:

[0177] The verification results of dilution linearity are shown in Table 21: Three dilution factors were investigated, namely 300-fold, 1200-fold, and 4800-fold. After correction by the dilution factor, the calculated average concentration of the quality control samples diluted 300-fold was -1.57% of the labeled value, with a precision of 7.60%; the calculated average concentration of the quality control samples diluted 1200-fold was 2.35% of the labeled value, with a precision of 1.15%; the calculated average concentration of the quality control samples diluted 4800-fold was -16.10% of the labeled value, with a precision of 7.85%. The verification results of dilution linearity from 300-fold to 4800-fold meet the requirements.

[0178]

[0179] Table 21: Summary of Dilution Linearity Experiment Data

[0180] Specificity:

[0181] The specificity verification results are shown in Table 22: The accuracy of ULOQ and LLOQ of the specific sample containing oseltamivir (1000ng / mL) solution was -13.25% and -10.53% respectively; the accuracy of ULOQ and LLOQ of the specific sample containing interferon α2b (2μg / mL) solution was -6.88% and -1.09% respectively; and the accuracy of ULOQ and LLOQ of the specific sample containing new crown S protein antibody (10000RU / mL) solution was -9.03% and -11.52% respectively; all met the requirements. The OD values ​​of the blank samples with relevant interfering substances added were all lower than the OD value of LLOQ. The specificity verification results of adding oseltamivir (1000ng / mL), interferon α2b (2μg / mL), and new crown S protein antibody (10000RU / mL) met the requirements.

[0182]

[0183]

[0184] Table 22: Summary of specific experimental data

[0185] Hemolysis selectivity:

[0186] The results of hemolysis selectivity validation are shown in Table 23: In the hemolysis sample prepared with hemolysis serum (2% V / V) diluted 100 times as the diluent, the LLOQ concentration sample accuracy deviation is -1.63%, the QH concentration sample accuracy deviation is -13.90%, the blank serum OD value is lower than the quantification lower limit OD value, and 100% of the sample accuracy is within ±20% (LLOQ is ±25%). And the detection OD value of the matrix without adding the analyte should be lower than the quantification lower limit OD value. The results of hemolysis selectivity validation meet the requirements.

[0187]

[0188] Table 23: Summary of hemolysis selectivity test data

[0189] The ELISA indirect method of the present invention detects the concentration of VZV-gE protein-specific IgG antibodies in human serum. After methodological verification, the standard curve and quantitative range, precision and accuracy, method durability, selectivity, specificity, dilution linearity and hook effect all meet the requirements for biological sample determination, and the verification results are all within an acceptable range;

[0190] The results showed that this method is suitable for the quantitative determination of VZV-gE protein-specific IgG antibody concentration in human serum. Under the experimental conditions, the stability sample prepared by spiking positive serum in blank serum was stable at room temperature for 24 hours, stable in a refrigerator at 2-8°C for 24 hours, stable after repeated freezing and thawing at -80°C for 6 times, and stable when frozen at -80°C for 164 days.

[0191] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for detecting IgG antibodies specific to varicella-zoster virus gE protein in human serum by high-sensitivity ELISA, characterized in that: The following steps are involved: Antigen coating: dilute the gE protein with a content of 2170 μg / mL to 0.5 μg / mL with 1× PBS phosphate buffer, add 100 μL / well to a 96-well plate, and keep at low temperature overnight; Washing the plate: Take out the ELISA plate coated overnight, discard the liquid in the well, pat dry, add 300μL / well of washing solution, let it stand and pat dry, repeat three times; Blocking: Add 300 μL / well of blocking solution, cover with sealing film and incubate; Repeat the plate washing process; Sample addition: Add 100 μL / well of the prepared quality control sample, cover with sealing film and incubate; Repeat the plate washing process; Add enzyme: add 100 μL / well of enzyme working solution, cover with sealing film and incubate; Repeat the plate washing process; Color development: Add TMB 100 μL / well and cover with sealing film to avoid light; Stop: Add 50 μL / well of stop solution and shake gently; Reading: Read the OD value at a wavelength of 450nm.

2. The method for detecting varicella-zoster virus gE protein-specific IgG antibodies in human serum by high-sensitivity ELISA as claimed in claim 1, characterized in that: The overnight temperature is 2-8℃.

3. The method for detecting varicella-zoster virus gE protein-specific IgG antibodies in human serum by high-sensitivity ELISA method as claimed in claim 2, characterized in that: Take out the ELISA plate coated overnight, discard the liquid in the wells, pat dry, add 300 μL / well of wash solution, let it stand and pat dry, the standing time is 1 min.

4. The method for detecting varicella-zoster virus gE protein-specific IgG antibodies in human serum by high-sensitivity ELISA as claimed in claim 3, characterized in that: 300 μL / well of blocking solution was added, and the plate was covered with a sealing film and incubated at 37°C and 400 rpm for 2 h.

5. The method for detecting varicella-zoster virus gE protein-specific IgG antibodies in human serum by high-sensitivity ELISA as claimed in claim 4, characterized in that: Add 100 μL / well of the prepared quality control sample, cover with sealing film and incubate at 37°C and 400 rpm for 1 hour.

6. The method for detecting varicella-zoster virus gE protein-specific IgG antibodies in human serum by high-sensitivity ELISA as claimed in claim 5, characterized in that: 100 μL / well of enzyme working solution was added, and the plate was covered with sealing film and incubated at 37°C and 400 rpm for 30 min.

7. The method for detecting varicella-zoster virus gE protein-specific IgG antibodies in human serum by high-sensitivity ELISA as claimed in claim 6, characterized in that: TMB 100 μL / well was added, and the plate was covered with a sealing film to protect from light. The color development was carried out in the dark at 37°C and 400 rpm for 15 min.

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